MOLECULAR ANALYSIS OF A NOVEL WINGED HELIX PROTEIN, WIN - EXPRESSION PATTERN, DNA-BINDING PROPERTY, AND ALTERNATIVE SPLICING WITHIN THE DNA-BINDING DOMAIN

We have cloned a novel winged helix factor, WIN, from the rat insulino ma cell line, INS-1. Northern blot analysis demonstrated that WIN is h ighly expressed in a variety of insulinoma cell lines and rat embryoni c pancreas and liver, In adults, WIN expression was detected in thymus , testis, lung, and several intestinal regions, We determined the DNA sequences bound in vitro by baculovirus-expressed WIN protein ire a po lymerase chain reaction-based selection procedure, WIN was found to bi nd with high affinity to the selected sequence 5'-AGATTGAGTA-3', which is similar to the recently identified HNF-6 binding sequence 5'-DHWAT TGAYTWWD-3' (where W = A or T, Y = T or C, H is not G, and D is not C) , We have isolated human WIN cDNAs by library screening and 5'-rapid a mplification of cDNA ends, Sequence analysis indicates that the carbox yl terminus of human WIN has been previously isolated as a putative ph osphorylation substrate, MPM2-reactive phosphoprotein 2 (MPP2); WIN ma y be regulated by phosphorylation, Alignment of the rat and human WIN cDNAs and their comparison with mouse genomic sequence revealed that t he WIN DNA binding domain is encoded by four exons, two of which (exon s 4 and 6) are alternatively spliced to generate at least three classe s of mRNA transcripts, These transcripts were shown by RNase protectio n assay to be differentially expressed in different tissues, Alternati ve splicing within the winged helix DNA binding domain might result in modulation of DNA binding specificity.